This research seeks to elucidate further the enzyme mechanisms involved in the assimilatory reduction of nitrate to ammonium. The significance of this pathway is evident since the various oxidized inorganic forms of nitrogen provide the ultimate nitrogen source for all life. Nitrate assimilation in Neurospora crassa is a two step process: the 2e minus reduction of nitrate to nitrite via nitrate reductase, followed by the 6e minus reduction of NO minus over 2 to ammonium by nitrite reductase. The nitrate reductase is an NADPH-specific, sulfhydryl-containing molybdoflavoprotein possessing a functional b-type cytochrome. The sequence of electron transfer has been established: NADPH yields FAD yields cytochrome by b557 yields MO yields NO minus over 3. Recent results using diazotized amino-pyridine adenine dinucleotide as an NADPH site-specific reagent suggest enzyme sulfhydryl groups mediate electron transfer between NADPH and FAD. The second enzyme, nitrite reductase, has been characterized by this laboratory as an NAD(P)H-dependent, FAD-requiring protein complex which mediates the stoichiometric reduction of nitrite to ammonium. Siroheme, an iron tetrahydroporphyrin, is a functional prosthetic group as judged by chemical, spectral and kinetic analysis of the enzyme. The proposed scheme of electron flow is: NAD(P)H yields FAD yields siroheme yields NO minus over 2. Dithionite can also serve as an electron donor to reduce nitrite in an FAD-independent reaction, and the complex also expresses an NAD(P)H-diaphorase activity which presumably does not involve the siroheme moiety. Primary objectives at this point include: (a) examination of these partial electron transport functions and the comparison of their characteristics of those of the overall NAD(P)H-nitrite reductase activity, and (b) purification of the nitrite reductase complex (MW 300,000) to homogeneity, and determination of its subunit organization. The integration of these results should provide insight into the relationships between the structure and function of the nitrite reductase.